The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit β₂-microglobulin (β₂m), and a peptide. Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2. Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/β₂m heterodimer. However, we identify two pathways for peptide dissociation from the heterotrimer:  (1) initial peptide dissociation leaving a heavy chain/β₂m heterodimer and (2) initial dissociation of β₂m, followed by peptide dissociation from the heavy chain. Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding. We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues. Thermodynamic and kinetic analyses of peptide−MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity. Finally, our data suggest a role for β₂m in dampening conformational dynamics in the heavy chain. Remaining conformational variability in the heavy chain once β₂m has bound may be a mechanism to promote promiscuity in peptide binding.